light-harvesting antennae is thought to play an important photoprotective role by mitigating oxi-
dative damage. Non-radiative energy dissipation at PSII is mediated by zeaxanthin and perhaps
also by antheraxanthin [9] and is proposed to occur at several sites within or around the PSII
reaction center [10]. Researchers have used differ- ent terms for energy dissipation, e.g. the quench-
ing
coefficient q
N
[11] versus
Stern – Volmer quenching [9,12] that is referred to as non-photo-
chemical quenching NPQ. In the most powerful source of AOS, chloro-
plasts, O
2 −
that is produced by photoreduction of O
2
at PSI and PSII is detoxified by the Mehler- peroxidase pathway [3,13]. The O
2 −
is reduced to hydrogen peroxide H
2
O
2
by superoxide dismu- tase SOD and then to H
2
O by ascorbate peroxi- dase APX, which is the key enzyme involved in
H
2
O
2
scavenging. These enzymes, together with monodehydroascorbate reductase, dehydroascor-
bate reductase, and glutathione reductase GR, constitute the major defense system against AOS
in the chloroplast [3,14]. Additionally, extraplastic H
2
O
2
quenching by peroxidase POD and cata- lase CAT is also increased in stress responses
[15]. A crucial role of these enzymes in protection against oxidative processes has been shown in
transgenic tobacco plants overexpressing either Mn-SOD or Fe-SOD [16,17]. In contrast, other
studies with transgenic plants suggest that en- hancement of a particular antioxidant enzyme
does not lead to increased protection [18,19].
Photooxidative damage is exacerbated by herbi- cides, which generate AOS either by direct in-
volvement in radical production or by inhibition of biosynthetic pathways [20,21], as well as atmo-
spheric pollutants and heavy metals [8]. Enhanced activities of antioxidants were associated with re-
sistance to herbicides such as paraquat and oxyfluorfen [22,23]. In the present study a potent
herbicide
norflurazon NF,
which blocks
carotenoid biosynthesis
by non-competitively
binding to phytoene desaturase [24], was used in leaves of Cucumis sati6us. It eliminates important
quenchers of the triplet chlorophyll Chl and
1
O
2
, thus initiating photooxidative processes. To assess
contribution of each inductive response of antioxi- dant to overall protective strategies to NF-caused
oxidative damage, photochemical efficiency of PSII, composition of photosynthetic pigments,
quenching parameters and activities of antioxidant enzymes were determined. The questions of
whether the level of photosynthetic photon flux density PPFD influences the NF-caused oxida-
tive stress was also examined, and, if so, whether the NF plants at different PPFDs have different
capacities to develop antioxidant responses.
2. Materials and methods
2
.
1
. Plant material and growth conditions Cucumber seeds C. sati6us L. cv Summer Long
were sown in vermiculite and transferred after 4 days into synthetic soil in plastic pots. Plants were
grown in a controlled environment growth cham- ber under a temperature of 25°C, a 16-h photope-
riod, and a light intensity of 200 mmol m
− 2
s
− 1
for 3 days. For NF treatment the 7-day-old plants were exposed in a surface application to 15 mM.
When the treatment was initiated the first leaves were about to emerge. Following NF application
plants were immediately returned to the growth chamber and exposed for 3 days under a 16-h
photoperiod with an irradiance of either low PPFD 30 mmol m
− 2
s
− 1
or high PPFD 300 mmol m
− 2
s
− 1
at 25°C. Inhibition of carotenoid biosynthesis causes a characteristic bleaching of
newly developed leaves. The four treatments em- ployed were: 1 CH, controlhigh PPFD; 2 NH,
NF treatmenthigh PPFD; 3 CL, controllow PPFD; and 4 NL, NF treatmentlow PPFD. The
first leaves were used for the measurements of Chl fluorescence, pigment contents and enzyme activi-
ties. The experiments were triplicated each with three determinations.
2
.
2
. Chl a fluorescence measurements In vivo Chl a fluorescence was measured after 5
min dark-adaptation at room temperature using a pulse amplitude modulation fluorometer PAM-
2000, Walz,
Effeltrich, Germany.
Minimal fluorescence yield, F
, was obtained upon excita- tion with a weak measuring beam from a pulse
light-emitting diode. Maximal fluorescence yield, F
m
, was determined after exposure to a saturating pulse of white light to close all reaction centers.
Determination of the quenching components q
P
and q
N
was conducted by the saturation pulse method and they were calculated as defined by
Schreiber et al. [25]. The quantum yield of electron transport through PSII Y = DFF
m
was calcu- lated according to Genty et al. [26]. Non-photo-
chemical fluorescence
quenching was
also quantified, as previously done by Bilger and
Bjo¨rkman [12] according to the Stern – Volmer equation, NPQ = F
m
F
m
− 1, where F
m
is the low- ered maximal yield during illumination with pho-
tosynthetically active radiation.
2
.
3
. Immunoblot analysis For the immunoblot of the extrinsic 33-kDa
protein of the oxygen-evolving complex in PSII reaction center, the method of Tae et al. [27] was
used. The thylakoid membranes isolated from chloroplasts were resuspended in 10 mM NaCl, 50
mM sucrose, and 50 mM sodium phosphate buffer, pH 7.4 and were sedimented at 10 000 × g
for 10 min. The pellets were resuspended in the same buffer as mentioned above. The chlorophylls
were removed with 80 acetone and the protein pellets were solubilized 1 SDS, 8 M urea, 1
2-mercaptoethanol, 10.7 mM phosphoric acid. The protein concentrations were measured with
the UV spectrophotometric method. Samples of protein to be blotted were electrophoresed in 12
SDS-polyacrylamide gel. The gel was run in 192 mM Glycine, 0.01 wv SDS, and 25 mM Tris –
HCl, pH 8.3. Polypeptides were transferred to nitrocellulose paper pore size: 0.45 mm; Hybond-
C, Amersham with a semi-dry transfer blotter 130-mA constant current, 60 min Model TE70,
Hoefer Scientific Instruments. The paper was washed in TBS buffer 500 mM NaCl, 20 mM
Tris – HCl, pH 7.4, incubated in a sealed plastic bag with 10 milk casein on a rocking shaker
RK1020 for 2 h, removed from the bag, and washed in TBS buffer. After incubating with the
antibody in TBS buffer containing 3 bovine serum albumin BSA for 2 h, and washing in
TTBS buffer containing 0.05 Tween-20, 500 mM NaCl, 20 mM Tris – HCl, pH 7.4, the paper was
again incubated with a second antibody [goat anti- rabbit IgG conjugated with horseradish peroxidase
Bio-Rad] in TBS containing 3 BSA. The paper was incubated on a rocking shaker for 1 h, washed
in TTBS buffer, and stained for 10 min with 0.017 4-chloro-1-naphthol and hydrogen perox-
ide in TBS buffer.
2
.
4
. Pigment extraction and analysis Extraction and HPLC anaysis of carotenoids
and Chls were done as described previously [28].
2
.
5
. Extraction of soluble protein Frozen leaves 0.25 g for CAT, GR, and APX;
0.5 g for POD and SOD were crushed to fine powder in a mortar under liquid N
2
. Soluble proteins were extracted by homogenizing the pow-
der in 2 ml of 100 mM potassium phosphate buffer, pH 7.5, containing 2 mM EDTA, 1
PVP-40, and 1 mM PMSF. For analysis of APX, the extraction buffer also contained 5 mM ascor-
bate. Insoluble material was removed by centrifu- gation at 15 000 × g for 20 min at 4°C, and the
supernatant was filtered through filter papers No. 1 Whatman, Maidstone, UK. Since maintenance
of consistent CAT electrophoretic mobility and GR activity was found to require the presence of
DTT, an aliquot of each sample was made to 10 mM DTT to be used for CAT and GR zy-
mograms and GR spectrometric assays. For the spectrophotometric assay of SOD, extracts were
passed through a PD-10 column Pharmacia, Upp- sala, Sweden.
2
.
6
. Enzyme assays CAT activity was determined by using a Clark-
type oxygen electrode Rank Brothers, Cambridge, UK according to the method of Natvig [29]. The
CAT assay was performed in a 3 ml volume containing N
2
-bubbled 50 mM potassium phos- phate buffer, pH 7.0, containing 20 mM H
2
O
2
. POD activity was determined specifically with gua-
iacol at 470 nm o = 25.2 mM cm
− 1
following the method of Egley et al. [30]. The reaction mixture
contained 40 mM potassium phosphate buffer pH 6.9, 1.5 mM guaiacol, and 6.5 mM H
2
O
2
in a 3-ml volume. SOD activity was determined as
described by Spychalla and Desborough [31]. The assay was performed at 25°C in a 3-ml volume
containing 50 mM Na
2
CO
3
NaHCO
3
buffer pH 10.2, 0.1 mM EDTA, 0.015 mM ferricytochrome
C, and 0.05 mM xanthine. APX activity was mea- sured spectrophotometrically by monitoring the
decline in A
290
as ascorbate o = 2.8 mM cm
− 1
was oxidized, using the method of Chen and As- ada [32]. The 3-ml reaction volume contained 100
mM potassium phosphate buffer pH 7.5, 0.5 mM ascorbate, and 0.2 mM H
2
O
2
at 25°C. GR activity was measured spectrophotometrically by measur-
ing the decline in A
340
as NADPH o = 6.2 mM cm
− 1
was oxidized, as described by Rao et al. [33]. The 3-ml assay mixture contained 100 mM
potassium phosphate buffer pH 7.8, 2 mM EDTA, 0.2 mM NADPH, 0.5 mM GSSG, and the
leaf extract. The assays were initiated by the addi- tion of NADPH at 25°C.
2
.
7
. Nati6e PAGE and acti6ity staining Equal amounts of protein from plants exposed
to different treatments were subjected to 10 non- denaturing polyacrylamide gels at 4°C for 1.5 h
with a constant current of 30 mA. After comple- tion of electrophoresis the gels were stained for the
enzymatic activities. Catalase activity was detected by incubating the gels in 3.27 mM H
2
O
2
for 25 min, rinsed in water, and stained in a solution of
1 potassium ferricyanide and 1 ferric chloride for 4 min [34]. Staining of POD isozymes was
achieved by incubating the gels in sodium citrate buffer, pH 5.0, containing 9.25 mM p-phenylene-
diamine and 3.92 mM H
2
O
2
for 15 min [35]. Gels were stained for SOD isoforms by soaking in 50
mM potassium phosphate, pH 7.8, containing 2.5 mM nitroblue tetrazolium in darkness for 25 min,
followed by soaking in 50 mM potassium phos- phate, pH 7.8, containing 28 mM nitroblue tetra-
zolium and 28 mM riboflavin in darkness for 30 min [33]. Gels were then exposed to light for
approximately 30 min. Following separation of APX, gels were soaked in 50 mM potassium phos-
phate buffer, pH 7.0, containing 2 mM ascorbate for 30 min [33]. The gels were incubated in the
same buffer containing 4 mM ascorbate and 2 mM H
2
O
2
for 20 min, and then soaked in 50 mM potassium phosphate buffer, pH 7.8, containing 28
mM tetramethyl ethylene diamine and 2.45 mM nitroblue tetrazolium for 15 min. Gels were
stained for GR activity in a solution of Tris – HCl, pH 7.5, containing 10 mg of 3-4,5-dimethylthia-
zol-2-4-2,5-diphenyl tetrazolium bromide, 10 mg of 2,6-dichlorophenolindophenol, 3.4 mM GSSG,
and 0.5 mM NADPH in darkness for 1 h [33].
3. Results
